Combined physical and immunotherapy for cancer

ABSTRACT

The present invention combines physical and immunologic therapies for the treatment of neoplasms by conditioning a targeted neoplasm with an immunoadjuvant (also called immuno-modulator or immunopotentiator) and then physically destroying the conditioned neoplasm. A number of physical therapies can be used to achieve the physical destruction of the conditioned tumor mass.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/720,685, filed Oct. 2, 1996, now U.S. Pat. No. 5,747,475which is a continuation-in-part of U.S. patent application Ser. No.08/416,158, filed Apr. 4, 1995. This application also claims the benefitof U.S. provisional application Ser. No. 60/041,327, filed Mar. 20,1997.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates generally to methods for treatingneoplasms and products derived therefrom and, more specifically, totreatment methods combining direct physical tumor therapy andimmunotherapy in order to induce immediate neoplastic cellulardestruction and simultaneously stimulate the self-immunological defensesystem against residual neoplastic cells.

2. Background

A neoplasm is an abnormal tissue that grows by cellular proliferationmore rapidly than normal. It continues to grow even after the stimulusthat initiated its growth dissipates. Neoplasms show a partial orcomplete lack of structural organization and functional coordinationwith the normal tissue and usually form a distinct mass which may beeither benign or malignant.

Cancer is a general term frequently used to indicate any of the varioustypes of malignant neoplasms, most of which invade surrounding tissues,may metastasize to several sites, and are likely to recur afterattempted removal and to cause death of the patient unless adequatelytreated. Cancer can develop in any tissue of any organ at any age.

Once an unequivocal diagnosis of cancer is made, treatment decisionsbecome paramount. Though no single treatment approach is applicable toall cancers, successful therapy must be focused on the primary tumor andits metastases, whether clinically apparent or microscopic.

Conventional Treatments

Historically, local and regional therapy, such as surgery or radiation,have been used in cancer treatment, along with systemic therapy, e.g.,drugs.

Surgery is the oldest effective form of cancer therapy. In 1988, about1,500,000 persons developed cancer; of those, about 515,000 had cancerof either the skin or cervix. About 985,000 had other systemic forms;64% had operable lesions, with an estimated cure rate of 62%. Cancersthat may be positively influenced with surgery alone, if detected inearly stages, include those of the cervix, breast, bladder, colon,prostate, larynx, endometrium, ovary, oral cavity, kidney, testis(nonsemino-matous) and lung (non-small cell). It must be noted, however,that the percentage rate of treatment success varies greatly between thecancer sites.

Radiation plays a key role in the remediation of Hodgkin's disease,nodular and diffuse non-Hodgkin's lymphomas, squamous cell carcinoma ofthe head and neck, mediastinal germ-cell tumors, seminoma, prostatecancer, early stage breast cancer, early stage non-small cell lungcancer, and medulloblastoma. Radiation can be used as palliative therapyin prostate cancer and breast cancer when bone metastases are present,in multiple myeloma, advanced stage lung and esophagopharyngeal cancer,gastric cancer, and sarcomas, and in brain metastases. Cancers that maybe curable with radiation alone include Hodgkin's disease, early-stagenon-Hodgkin's lymphomas, cancers of the testis (seminomal), prostate,larynx, cervix, and, to a lesser extent, cancers of the nasopharynx,nasal sinuses, breast, esophagus, and lung.

Antineoplastic drugs are those that prevent cell division (mitosis),development, maturation, or spread of neoplastic cells. The idealantineoplastic drug would destroy cancer cells without adverse effectsor toxicities on normal cells, but no such drug exists. Despite thenarrow therapeutic index of many drugs, however, treatment and even cureare possible in some patients. Certain stages of choriocarcinoma,Hodgkin's disease, diffuse large cell lymphoma, Burkitt's lymphoma andleukemia have been found to be susceptible to antineoplastics, as havebeen cancers of the testis (nonseminomatous) and lung (small cell).Common classes of antineoplastic drugs include alkylating agents,antimetabolites, plant alkaloids, antibiotics, nitrosoureas, inorganicions, enzymes, and hormones.

Despite some success, the above treatments are not effective to thedegree desired, and the search has continued for more efficacioustherapies.

Recent Advances

Two of the more recent oncological treatment modalities investigated bythe medical community are photodynamic therapy and tumor immunotherapy.

I. Photodynamic Therapy

It has been known for many years that photosensitizing compounds show aphotochemical reaction when exposed to light. Photodynamic therapy (PDT)uses such photosensitizing compounds and lasers to produce tumornecrosis. Treatment of solid tumors by PDT usually involves the systemicadministration of tumor localizing photosensitizing compounds and theirsubsequent activation by laser. Upon absorbing light of the appropriatewavelength the sensitizer is converted from a stable atomic structure toan excited state. Cytotoxicity and eventual tumor destruction aremediated by the interaction between the sensitizer and molecular oxygenwithin the treated tissue to generate cytotoxic singlet oxygen.

Two good general references pertaining to PDT, biomedical lasers andphotosensitizing compounds, including light delivery and dosageparameters, are Photosensitizing Compounds: Their Chemistry, Biology andClinical Use, published in 1989 by John Wiley and Sons Ltd., Chichester,U.K., ISBN 0 471 92308 7, and Photodynamic Therapy and BiomedicalLasers: Proceedings of the International Conference on PhotodynamicTherapy and Medical Laser Applications, Milan, Jun. 24-27 1992,published by Elsevier Science Publishers B.V., Amsterdam, TheNetherlands, ISBN 0 444 81430 2, both incorporated herein by reference.

United States patents related to PDT include U.S. Pat. Nos. 5,095,030and 5,283,225 to Levy et al.; U.S. Pat. No. 5,314,905 to Pandey et al.;U.S. Pat. No. 5,214,036 to Allison et al; and U.S. Pat. No. 5,258,453 toKopecek et al., all of which are incorporated herein by reference. TheLevy patents disclose the use of photosensitizers affected by awavelength of between 670-780 nm conjugated to tumor specificantibodies, such as receptor-specific ligands, immunoglobulins orimmunospecific portions of immunoglobulins. The Pandey patents aredirected to pyropheophorbide compounds for use in standard photodynamictherapy. Pandey also discloses conjugating his compositions with ligandsand antibodies. The Allison patent is similar to the Levy patents inthat green porphyrins are conjugated to lipocomplexes to increase thespecificity of the porphyrin compounds for the targeted tumor cells. TheKopecek patent also discloses compositions for treating canceroustissues. These compositions consist of two drugs, an anti-cancer drugand a photoactivatable drug, attached to a copolymeric carrier. Thecompositions enter targeted cells by pinocytosis. The anti-cancer drugacts after the targeted cell has been invaded. After a period of time, alight source is used to activate the photosensitized substituent.

II. Tumor Immunotherapy

The major functions of the immune system are to develop the concept of“self” and eliminate what is “nonself”. Although microorganisms are theprincipal nonself entities encountered every day, the immune system alsoworks to eliminate neoplasms and transplants. See Chapters 18 and 103 ofThe Merck Manual of Diagnosis and Therapy, Sixteenth Edition, publishedin 1992 by Merck Research Laboratories of Rahway, N.J., ISBN0911910-16-6 and 0076-6526; the same being incorporated herein byreference.

There are several distinct types of immunity. Nonspecific, or innate,immunity refers to the inherent resistance manifested by a species thathas not been immunized (sensitized or allergized) by previous infectionor vaccination. Its major cellular component is the phagocytic system,whose finction is to ingest and digest invading microorganisms.Phagocytes include neutrophils and monocytes in the blood andmacrophages in the tissues. Complement proteins are the major solublecomponent of nonspecific immunity. Acute phase reactants and cytokines,such as interferon, are also part of innate immunity.

Specific immunity is an immune status in which there is an alteredreactivity directed solely against the antigenic determinants(infectious agent or other) that stimulated it. It is sometimes referredto as acquired immunity. It may be active and specific, as a result ofnaturally acquired (apparent or inapparent) infection or intentionalvaccination; or it may be passive, being acquired from a transfer ofantibodies from another person or animal. Specific immunity has thehallmarks of learning, adaptability, and memory. The cellular componentis the lymphocyte (e.g., T-cells, B-cells, natural killer (NK) cells),and immunoglobulins are the soluble component.

The action of T-cells and NK-cells in recognizing and destroyingparasitized or foreign cells is termed cell-mediated immunity. Incontradistinction to cell-mediated immunity, humoral immunity isassociated with circulating antibodies produced, after a complexrecognition process, by B-cells.

As regards tumor immunology, the importance of lymphoid cells in tumorimmunity has been repeatedly shown. A cell-mediated host response totumors includes the concept of immunologic surveillance, by whichcellular mechanisms associated with cell-mediated immunity destroy newlytransformed tumor cells after recognizing tumor-associated antigens(antigens associated with tumor cells that are not apparent on normalcells). This is analogous to the process of rejection of transplantedtissues from a nonidentical donor. In humans, the growth of tumornodules has been inhibited in vivo by mixing suspensions of a patient'speripheral blood lymphocytes and of tumor cells, suggesting acell-mediated reaction to the tumor. In vitro studies have shown thatlymphoid cells from patients with certain neoplasms show cytotoxicityagainst corresponding human tumor cells in culture. These cytotoxiccells, which are generally T-cells, have been found with neuroblastoma,malignant melanomas, sarcomas, and carcinomas of the colon, breast,cervix, endometrium, ovary, testis, nasopharynx, and kidney. Macrophagesmay also be involved in the cell-mediated host's response to tumors whenin the presence of tumor-associated antigens, lymphokines or interferon.

Humoral antibodies that react with tumor cells in vitro have beenproduced in response to a variety of animal tumors induced by chemicalcarcinogens or viruses. Hydridoma technology in vitro permits thedetection and production of monoclonal antitumor antibodies directedagainst a variety of animal and human neoplasms. Antibody-mediatedprotection against tumor growth in vivo, however, has been demonstrableonly in certain animal leukemias and lymphomas. By contrast, lymphoidcell-mediated protection in vivo occurs in a broad variety of animaltumor systems.

Immunotherapy for cancer is best thought of as part of a broadersubject, namely biologic therapy, or the administration ofbiologic-response modifiers. These agents act through one or more of avariety of mechanisms (1) to stimulate the host's antitumor response byincreasing the number of effector cells or producing one or more solublemediators; (2) to serve as an effector or mediator; (3) to decrease hostsuppressor mechanisms; (4) to alter tumor cells to increase theirimmunogenicity or make them more likely to be damaged by immunologicprocesses; or (5) to improve the host's tolerance to cytotoxics orradiation therapy. Heretofore the focus of cell-mediated tumorimmunotherapy has been on reinfusion of the patient's lymphocytes afterexpansion in vitro by exposure to interleukin-2. One variation includesisolating and expanding populations of lymphocytes that have infiltratedtumors in vivo, so-called tumor-infiltrating lymphocytes. Another is theconcurrent use of interferon, which is thought to enhance the expressionof histocompatibility antigens and tumor-associated antigens on tumorcells, thereby augmenting the killing of tumor cells by the infusedeffector cells.

Humoral therapy, on the other hand, has long concentrated on the use ofantitumor antibodies as a form of passive immunotherapy, in contrast toactive stimulation of the host's own immune system. Another variation isthe conjugation of monoclonal antitumor antibodies with toxins, such asricin or diphtheria, or with radioisotopes, so the antibodies willdeliver these toxic agents specifically to the tumor cells. Activeimmunization with a host's own tumor cells, after irradiation,neuraminidase treatment, hapten conjugation, or hybridization has alsobeen tried. Clinical improvement has been seen in a minority of patientsso treated. Tumor cells from others have been used after theirirradiation in conjunction with adjuvants in acute lymphoblasticleukemia and acute myeloblastic leukemia after remission. Prolongationof remissions or improved reinduction rates have been reported in someseries, but not in most. Interferons, tumor necrosis factor andlymphotoxins have also been used to affect immunologically mediatedmechanisms. A recent approach, using both cellular and humoralmechanisms, is the development of “heterocross-linked antibodies,”including one antibody reacting with the tumor cell linked to a secondantibody reacting with a cytotoxic effector cell, making the latter morespecifically targeted to the tumor. Host immune cell infiltration into aPDT treated murine tumor has been reported.

Combined PDT and Immunotherapy

The potential for combining PDT with immunotherapy was explored byKrobelik, Krosl, Dougherty and Chaplin. See Photodynamic Therapy andBiomedical Lasers, supra, at pp. 518-520. In their study, theyinvestigated a possibility of amplification of an immune reaction to PDTand its direction towards more pervasive destruction of treated tumors.The tumor, a squamous cell carcinoma SCCVII, was grown on female C3Hmice. An immunoactivating agent SPG (a high molecular weight B-glucanthat stimulates macrophages and lymphoid cells to become much moreresponsive to stimuli from cytokines and other immune signals) wasadministered intramuscularly in 7 daily doses either ending one daybefore PDT or commencing immediately after PDT. Photofrin based PDT wasemployed; photofrin having been administered intravenously 24 hoursbefore the light treatment. The SPG immunotherapy was shown to enhancethe direct killing effect of the PDT. The indirect killing effect (seenas a decrease in survival of tumor cells left in situ) was, however,much more pronounced in tumors of animal not receiving SPG. Thedifference in the effectiveness of SPG immunotherapy when performedbefore and after PDT suggested that maximal interaction is achieved whenimmune activation peaks at the time of the light delivery or immediatelythereafter. With SPG starting after PDT (and attaining an optimal immuneactivation 5-7 days later), it is evidently too late for a beneficialreaction.

In another study the use of PDT to potentiate the effect of bioreactivedrugs that are cytotoxic under hypoxic conditions was investigated. SeePhotodynamic Therapy and Biomedical Lasers, supra, at pp. 698-701. Itwas found that the antitumor activity of such drugs can be enhanced invivo when they are used in combination with treatments that increasetumor hypoxia.

In application Ser. No. 08/416,158, filed Apr. 4, 1995 forLASER/SENSITIZER ASSISTED IMMUNOTHERAPY, the inventors disclosedphotophysically destroying a tumor while at the same time generating anin situ autologous vaccine to provide a long term humoral immunityagainst neoplastic cellular multiplication. The photothermal destructionof the tumor also initiates a host immune response. An in situ vaccineis formed when photothermal destruction occurs, as the fragmented tissueand cellular molecules are dispersed within the host in the presence ofan immunoadjuvant. The self-immunological defense system is stimulatedwhen this mixture of materials circulates in the host and is detected bythe immunological surveillance system. There follows an immediatemobilization of cell mediated immunity, which shifts to a humoralimmunity with the production of cytotoxic antibodies over time. Thus, asignificant improvement was made in the treatment of cancer whereby thepatient is provided with not only immediate tumor destruction but alsowith the ability to protect the body against a proliferation of residualor metastatic neoplastic cells.

Application Ser. No. 08/720,685, filed Oct. 2, 1996 for CHITOSAN DERIVEDBIOMATERIALS is drawn to novel chitosan-derived biomaterials and theirbiomedical uses, glycated chitosan being the preferred immunoadjuvantused laser/sensitizer assisted immunotherapy.

Application Ser. Nos. 08/416,158 and 08/720,685 are hereby incorporatedherein by reference.

Object

It is an object of this invention to further improve the treatment ofneoplasms by providing additional procedures that combine direct tumorablation and immunotherapy in order to induce immediate neoplasticcellular destruction and simultaneously stimulate the self-immunologicaldefense system against residual neoplastic cells.

It is a further object of the invention to provide methods forgenerating an in situ autologous vaccine and for deriving productsuseful in cancer related diagnostic and therapeutic procedures.

SUMMARY OF THE INVENTION

Laser/sensitizer assisted immunotherapy encompasses introducing into aneoplasm (1) a chromophore and (2) an immunoadjuvant and then lasing theneoplasm at an irradiance sufficient to induce neoplastic cellulardestruction and to stimulate cell-mediated and humoral immune responses.

The present invention is directed to various means of inducingneoplastic cellular destruction while still achieving stimulation ofcell-mediated and humoral immune responses.

In accordance with one aspect of the present invention, animmunoadjuvant is introduced into the targeted tumor to obtain aconditioned tumor and the conditioned tumor is ablated by heat. Thehyperthermia may be generated by lasers, ultrasound, microwaves,radiofrequency induction or electric currents.

In another embodiment the conditioned tumor is destroyed by cryotherapy.

Thus, a number of techniques, invasive and non-invasive, are used toinduce neoplastic cellular destruction of a tumor conditioned with animmunoadjuvant in order to achieve direct killing of the tumor mass andobtain cell-mediated and humoral immune responses.

The present invention has several advantages over other conventional andunconventional treatment modalities. The most significant advantage is acombined acute and chronic tumor destruction. The acute tumor loss iscaused by ablation of the neoplastic tissue, on a large and controlledscale, in the immediate area, reducing the tumor burden and hence thebase of multiplication so that the self-defense system can fight aweaker enemy. When this direct destruction occurs, the fragmented tissueand cellular molecules are disbursed within the host in the presence ofthe immunologically potentiating material, such as chitosan. In effect,an in situ vaccine is formed. This mixture of materials then circulatesin the host and is detected by the immunological surveillance system.There follows an immediate mobilization of cell-mediated immunity whichencompasses NK-cells and recruited killer T-cells. These cells migrateto the sites of similar antigens or chemicals. In time, thecell-mediated immunity shifts to a humoral immunity with the productionof cytotoxic antibodies. These antibodies freely circulate about thebody and attach to cells and materials for which they have been encoded.If this attachment occurs in the presence of complement factors, theresult is cellular death. The time frames for these two immunologicmodes of action are 0 to 2 weeks for the cell-mediated response, whilethe humoral arm matures at approximately 30 days and should persist forlong periods, up to the life span of the host.

With the present invention collateral damage is reduced to a tolerablelevel. If a laser is used, the laser power is carefully chosen under acertain damage threshold so that the laser will do little damage totissue in the path of the laser beam, such as skin. This characteristicmakes a non-invasive treatment possible. Even in the case where diseasedtissues are deep inside the body, an endoscope and fiber optics caneasily reach the treatment site.

A chromophore of a complementary absorption wavelength makes lasertreatment highly selective. Only the chromophore injected area sustainsnoticeable tissue damage. The concentration of chromophore, the dosageof chromophore and immunoadjuvant, and the timing of administrationallow for temporal and spatial control of the induced photothermaleffect. The optimal administration can be achieved by considering thephysical and chemical characteristics of the chromophore and byconsidering the tissue responses to the photothermal interaction.Equally important are the natural reactions between the chromophore andits host tissues without any laser stimulation, such as the moleculesbreaking down over time, as well as the migration of molecules throughthe circulatory and excretory systems. The preferred chromophore of thepresent invention, ICG, is non-toxic and can be easily excreted in ashort period through the liver and kidney.

In sum, long term survival with total cancer eradication can be achievedby the present invention. It is a combined result of reduced tumorburden due to direct ablation of the tumor mass and an enhanced immunesystem response due to conditioning the tumor with chitosan or otherimmunomodulators prior to destroying the tumor.

Still other objects and advantages of the present invention will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein there is shown and described only thepreferred embodiments of the invention, simply by way of illustration ofthe best mode contemplated for carrying out the invention. As will berealized, the invention is capable of modifications in various obviousrespects, all without departing from the invention. Accordingly, thedescription should be regarded as illustrative in nature, and not asrestrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart related to a study of murine mammary tumors showingtumor burden over time for a particular murine subject whose primarytumor was treated using laser/sensitizer assisted immunotherapy.

FIG. 2 is a chart similar to FIG. 1 for a second treated murine subject.

FIG. 3 is a chart showing tumor burden over time for untreated secondarytumors in the murine subject of FIG. 1.

FIG. 4 is a chart showing tumor burden over time for untreated secondarytumors in the murine subject of FIG. 2.

FIG. 5 is a chart showing tumor burden over time for three other treatedmurine subjects.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention contemplates combining physical and immunologictherapies for the treatment of neoplasms by conditioning a targetedneoplasm with an immunoadjuvant (also called immuno-modulator orimmunopotentiator) and then physically destroying the conditionedneoplasm. A number of techniques or procedures can be used to achievethe physical destruction of the conditioned tumor mass.

A. Laser/Sensitizer Assisted Immunotherapy

Laser/sensitizer assisted immunotherapy was first described in U.S.patent application Ser. No. 08/416,158. In this modality the targetedtumor is conditioned with an immunoadjuvant, preferably glycatedchitosan, and a chromophore and is then lased, preferably in anon-invasive manner, with a laser having a wavelength of absorptioncorresponding to that of the chromophore.

The chromophore and immunoadjuvant are preferably combined into asolution for injection into the center of the tumor mass. It should berecognized however that other methods may be sufficient for localizingthe chromophore and immunoadjuvant in the tumor site. One suchalternative delivery means is conjugation of the chromophore orimmunoadjuvant or both to a tissue specific antibody or tissue specificantigen, such that delivery to the tumor site is enhanced. Any onemethod, or a combination of varying methods, of localizing thechromophore and immunoadjuvant in the tumor site is acceptable so longas the delivery mechanism insures sufficient concentration of thecomponents in the neoplasm.

Chromophore

The selection of an appropriate chromophore is largely a matter ofcoordination with an acceptable laser wavelength of radiation. Thewavelength of radiation used must, of course, be complementary to thephotoproperties (i.e., absorption peak) of the chromophore. Otherchromophore selection criteria include ability to create thermal energy,to evolve singlet oxygen and other active molecules, or to be toxic intheir own right such as cis-platinin. In the present invention, thepreferred wavelength of radiation is 808±10 nm. The desired chromophoreshave strong absorption in the red and near-infrared spectral region forwhich tissue is relatively transparent. Another advantage of thiswavelength is that the potential mutagenic effects encountered withUV-excited sensitizers are avoided. Nevertheless, wavelengths of between150 and 2000 nm may prove effective in individual cases. The preferredchromophore is indocyanine green. Other chromophores may be used,however, their selection being based on desired photophysical andphotochemical properties upon which photosensitization efficiency andphotocytotoxicity are dependent. Examples of alternative chromophoresinclude, but are not limited to, methylene blue, DHE(polyhaematoporphrin ester/ether), m-THPP(tetra(meta-hydroxyphenyl)porphyrin), AlPcS₄ (aluminium phthalocyaninetetrasulphonate), ZnET2 (zinc aetio-purpurin), and Bchla(bacterio-chlorophyll a).

Glycated Chitosan

The preferred immunomodulator is chitosan. Chitosan is a derivative ofchitin, a compound usually isolated from the shells of some crustaceanssuch as crab, lobster and shrimp. Chitin is a linear homopolymercomposed of N-acetylglucosamine units joined by β1→4 glycosidic bonds.Chitin, chitosan (partially deacetylated chitin) and their derivativesare endowed with interesting chemical and biological properties thathave led to a varied and expanding number of industrial and medicalapplications.

The presence of primary and secondary alcohol groups, and of primaryamino groups in chitosan, facilitate a number of approaches for chemicalmodifications designed mainly to achieve their solubilization and toimpart special properties for specific applications. Theirbiodegradability and lack of toxicity renders them “biologicallyfriendly,” since their degradation products can be utilized for thebiosynthesis of glycoconjugate components of living tissues. Chitosanand its derivatives have been utilized for bandages and sutures, burndressings, skin substitutes, bone and dental prostheses, food packaging,drug encapsulation, cosmetics, metal chelation and associatedantioxidant effects, waste water treatment, hemostasis, anticoagulants(after sulfation), and dye doping, among other things.

Solubilization of chitin and chitosan can be achieved by partialhydrolysis to oligosaccharides. For chitosan, treatment with a varietyof acids, both organic and inorganic, leads to the formation of watersoluble chitosonium salts by protonation of the free amino groups.Additional modifications of the amino groups include the introduction ofchemical groups such as carboxymethyl, glyceryl, N-hydroxybutyl andothers. Glycation, i.e., non-enzymatic glycosylation of the free aminogroups of chitosan, followed by stabilization by reduction, offers anovel approach for the preparation of the chitosan gels and solutionsutilized in the present invention.

Glycated chitosan, as indicated above, refers to the products resultingfrom the reaction between the free amino groups of chitosan and thecarbonyl groups of reducing monosaccharides and/or oligosaccharides. Theproducts of this reaction (mainly a mixture of Schiff bases, i.e. thecarbon atom of the carbonyl group double bonded to the nitrogen atom ofthe amino group, and Amadori products, i.e. the carbon atom of saidcarbonyl group bonded to the nitrogen atom of said amino group by asingle bond while an adjacent carbon atom is double bonded to an oxygenatom) may be used as such or after stabilization by reduction withhydrides, such as sodium borohydride, or by exposure to hydrogen in thepresence of suitable catalysts. The galactose derivative of chitosan isparticularly preferred insofar as it has a relatively higher naturallyoccurring incidence of its straight chain form. The glycated chitosanmay be prepared in a powder form, as a viscous suspension, or in otherforms.

One protocol for the preparation of glycated chitosan for use in thepresent invention is as follows: 3 grams of a reducing monosaccharide(e.g., glucose, galactose, ribose), or an equivalent amount of areducing oligosaccharide, is dissolved in 100 ml of distilled waterunder gentle magnetic stirring in an Erlenmeyer flask. One gram ofchitosan is added. When the suspension is homogeneous, 0.25 ml oftoluene is added, and the flask is sealed with aluminum foil. Themagnetic stirring continues for 24 hours at room temperature. Afterstirring, the suspension is placed in a ventilated fume hood where 1.327grams of sodium borohydride in 5 ml of 0.1M sodium hydroxide is added toreduce Schiff bases and Amadori products. The solution is then coveredloosely with foil, stirred for 10 minutes at room temperature and 50minutes in an ice bath. After this stirring step, the flask is removedfrom the ice bath and the solution is acidified to a pH of 5.5 by thedropwise addition of glacial acetic acid (approximately 1.9 ml) underfurther magnetic stirring to decompose excess borohydride. The solutionis then centrifuged for 15 minutes at 15,000 rpm (on a Sorval, rotorSS-34, at 4° C.) in five glass (Corex) centrifuge tubes. The supernatantis decanted and the clear gel layer overlaying the pellets is gentlyscraped with a steel spatula. Using the supernatant from two of thecentrifuge tubes, the pellets are resuspended and recentrifuged in twotubes. Again, the supernatant is decanted and the gel is collected asabove. The combined pellets are centrifuged a third time after beingresuspended in the supernatant of one tube. The gel is again collectedafter decanting the supernatant. Pooled gel is then dispersed in pooledsupernatant to obtain a homogeneous suspension, which is placed in threedialysis bags (Spectrapor, 25 mm flat width, 12,000-14,000 mol. wt.cut-off). The suspensions are dialyzed overnight at 4° C. against 3.5gallons of distilled water. The bags are then placed in fresh distilledwater and dialysis is continued for an additional 7 hours. Afterdialysis, the dialysate is removed from the bags and it is homogenizedby 3 bursts (10 seconds each) in a Waring blender at high speed. Theresulting viscous solution is stored frozen. Before use in the presentinvention, the frozen material is thawed in a water bath at 37° C., thenmixed in a Waring blender to achieve a homogeneous mix.

Alternatively, 100 ml of 1.0% (by weight of chitosan) galactosederivative of chitosan could be prepared as follows:

1. 250 μl of glacial acetic acid is added to 100 ml of pure water andthe mixture is stirred.

2. 1.00 grams (by dry weight) of chitosan is added. Stirring continuesuntil all of the chitosan dissolves (there will be a few pieces ofinsoluble non-chitosan impurities). This will take 1-2 hours.

3. 3.00 grams of galactose is added. Stir and let react for 16 hours ormore.

4. It should be noted that hydrogen is produced in this step. Whilevigorously stirring, a 10% sodium borohydride solution (1.0 gram ofsodium borohydride dissolved in 0.1N sodium hydroxide brought to 10.0 mlvolume) is added while monitoring the pH and the liquid/foam level inthe container. When the pH approaches 6.0, 250 μl of glacial acetic acidis added to lower the pH. The sodium borohydride is added and the pHadjusted until 5.0 ml of the 10% sodium borohydride has been added andthe pH is 5.3-5.8. This will require approximately 750 μl of acetic acidand take about 2 hours.

5. The liquid/foam produced is transferred to centrifuge tubes and iscentrifuged for 10 minutes at moderate g (approx. 1500) to break up thefoam and separate the particulates. Centrifugation is repeated asnecessary (½-1 hour).

6. The supernatant liquid is then transferred to dialysis tubing(Spectrapor 1) and dialysis is conducted against 4 liters of pure waterfor 4 hours.

7. To remove excess water acquired during dialysis, the tubing is placedunder an air stream (keeping the membrane moist on the inside) for aperiod of time (2-6 hours) until the weight is reduced the requiredamount. (20.4 mm diameter tubing loses approximately 6 g/hour in a fumehood doorway).

8. The dialysis is repeated in 4 liters of water for 6 hours. Then theweight is readjusted in accordance with the previous step.

9. Final dialysis is conducted against 16 liters of pure water for 16hours, then adjust weight or lyophilize to dryness.

The structure of galacto-chitosan and the preparation ofgalacto-chitosan from chitosan and D-galactose by reductive ammunitionis shown below.

Formula Biopolymer Structure of galactochitosan (GC) Structure

Molecular Weight (approx.) 1.5 million Preparation of Galactochitosan(GC)

Use of Glycated Chitosan as an Immunoadjuvant

In the preferred embodiment, ICG powder is blended with the glycatedchitosan preparation to yield a solution having a concentration ofbetween 0.1% to 2% (0.1 to 2 grams/100 ml) of ICG to glycated chitosansolution. The solution is kept warm until use. The effective dosage ofthe solution ranges from 70 to 2000 μl. A 100 ml solution of chromophoreand glycated chitosan immunoadjuvant also can be prepared from powderedforms of both constituents by added 0.25 grams of ICG and 0.5 grams ofglycated chitosan to 100 ml of pure water to yield a useful solutionhaving 0.25% ICG and 0.5% glycated chitosan by weight. Alternatively,ICG in solution can be mixed in appropiate stoichiometric amounts withglycated chitosan solutions to achieve preferred compositions.

As for laser parameters, a solid state diode laser that emits light in acontinuous wave through fiber optics of a diameter between 100 and 2000μm is preferred, although other lasers may be used, including banks ofindividual lasers that may or may not all be of the same wavelength. Thelaser power used can vary between 1 and 60 watts, the preferred powerbeing between 1 and 5 watts. The irradiance duration should last between1 and 60 minutes, 5 to 15 minutes being favored. The temperature of thelased tumor mass should preferably be raised to about 140° F. or 60° C.

In the most preferred embodiment, a solution of ICG and glycatedchitosan is prepared as described above at a concentration of 0.25 to 2%of ICG to chitosan. The solution is injected into the center of theneoplasm at a dosage of 70 to 1000 μl. The neoplasm is then lased usinga laser preferably having a power of about 5 watts and a wavelength ofradiation capable of readily penetrating normal cellular tissues withoutsignificant disruption. The irradiation preferably continues for aduration sufficient to elevate the temperature of the neoplasm to alevel that induces neoplastic cellular destruction and liberates tumorantigens which stimulate cell-mediated and humoral immune responses.

Other Uses for Glycated Chitosan and Derivatives Thereof

Other medical and industrial uses for glycated chitosan are anticipated.In addition to its use as an immunoadjuvant or a component thereof incombination with sensitizing dyes and laser/sensitizer assistedimmunotherapy, glycated chitosan might be used to facilitate theapplication of sodium fluoride to the surface of teeth prior to fusiononto the enamel by laser irradiation. It might also be used to preparesuspensions of hydroxyapatite and other formulations of calciumphosphate utilized for bone and dental prosthesis. Another use would beas a component of skin substitutes, sutures, dressings and bandages forburns, wounds and surgical procedures.

A further use of glycated chitosan, alone or in combination with otherdrugs, might be as an antiinfection treatment in septicemia, antibioticresistance, or antibiotic intolerance. It might also be used in cosmeticand pharmaceutical formulations, creams, salves, etc.

A still further use would be as an immunostimulant in the treatment ofimmuno-compromised patients including but not limited to cancer andacquired immunodeficiency syndrome. This includes, but is not limitedto, the use of chitosan derivatives with side chains derived from tumorsand microorganisms.

Gel and soluble forms of glycated chitosan will be used individually orin combination, both as such and/or after additional chemical orenzymatic modification. These modifications include, but are not limitedto, the generation of reactive groups such as carbonyls and carboxylgroups on the substituents introduced by glycation.

Aldehydes will be generated by oxidation of the carbohydrate side chain(e.g. treatment with periodate or lead tetraaceate) or, for example, theenzymatic oxidation of the primary alcohol group of galactosyl residueswith galactosyl oxidase.

Oxidation of the aldehyde groups (e.g. by treatment with hypohalites)will be utilized to obtain the carboxylic acid derivatives.Alternatively, bifunctional compounds containing both free carbonyl andcarboxylic groups (e.g. uronic acids) will be utilized during theglycation reaction.

Chitosan deamination with nitrous acid generates reducing aldoses andoligosaccharides suitable for the glycation of chitosan. Deamination ofthe deacetylated glucosaminyl residues by nitrous acid results in theselective cleavage of their glycosidic bonds with the formation of2,5-anhydro-D-mannose residues. Depending on the composition of specificareas of the chitosan chain, the anhydro hexose could be released as themonosaccharide, or occupy the reducing end of an oligosaccharide.Release of free N-acetylglucosamine could also occur from some regionsof the chitosan chain. Similar treatment of N-deacetylated glycoproteinsand glycolipids can be utilized to obtain oligosaccharides of definedchemical composition and biological activity for special preparations ofglycated chitosan. This includes normal as well as pathologicalglycoconjugates.

The various products obtained by chitosan glycation will be utilized assuch or reacted with other natural or synthetic materials, e.g.,reaction of aldehyde-containing derivatives of glycated chitosan withsubstances containing two or more free amino groups, such as on the sidechains of amino acids rich in lysine residues as in collagen, onhexosamine residues as in chitosan and deacetylated glycoconjugates, oron natural and synthetic diamines and polyamines. This is expected togenerate crosslinking through Schiff base formation and subsequentrearrangements, condensation, dehydration, etc.

Stabilization of modified glycated chitosan materials can be made bychemical reduction or by curing involving rearrangements, condensationor dehydration, either spontaneous or by incubation under variousconditions of temperature, humidity and pressure.

The chemistry of Amadori rearrangements, Schiff bases and theLeukart-Wallach reaction is detailed in The Merck Index, Ninth Edition(1976) pp. ONR-3, ONR-55 and ONR-80, Library of Congress Card No.76-27231, the same being incorporated herein by reference. The chemistryof nucleophilic addition reactions as applicable to the presentinvention is detailed in Chapter 19 of Morrison and Boyd, OrganicChemistry, Second Edition (eighth printing 1970), Library of CongressCard No. 66-25695, the same being incorporated herein by reference.

Experimental Studies

Further description of laser/sensitizer assisted immunotherapy utilizinga chitosan-derived immunoadjuvant, including components, parameters andprocedures, is contained in the following summary of a study of murinemammary tumors.

Materials and Methods

1. The Laser

A diode laser was used in this study. The Industrial Semiconductor LaserISL50F (McDonnell Douglas Aerospace, St. Louis, Mo.), employs a diodearray that is electrically powered by a diode driver. The laser emitsradiation at wavelength of 808±10 nm, either in pulsed or in continuousmode. Its maximum near infrared power in the available configurationoutput is 35 watts. The laser is operated with standard electric power(110/120 VAC). A microprocessor monitors and adjusts the laser operatingparameters. Laser energy can be delivered through optical fibers ofvarious sizes. A red laser diode emits 670 nm light at 0.9 mW as theaiming beam. The continuous wave mode was employed in this experiment.

In the experiment, different power and duration of laser irradiation,ranging from 3 minutes at 5 watts to 5 minutes at 15 watts, were used.The output power was measured before, during and after the procedureusing a Joule/Watt meter (Ophir Optics, Israel). Two types of fiberoptics were used: 600 μm and 1200 μm in diameter.

2. The Preparation of Animals

Wistar Furth female rats, age 6 to 7 weeks and weighing 100 to 125grams, were chosen for the study. The model is a metastatictransplantable rat mammary tumor. The tumor strain was the DMBA-4. Tumorcells (about 25,000 cells) were transplanted to the rats by injectioninto the superficial inguinal area between the skin and the musclelayer. Certain selected rats were injected with tumor cells in both leftand right inguinal areas for simultaneous tumor growth.

The rats were fed a special food, a high saturated fat diet, tofacilitate the growth of the tumor. The tumor usually grew to about 1 to4 cm³ within 10 to 14 days of tumor transplantation. In most cases therat tumors were treated before they grew beyond 5 cm³.

Before laser treatment, anesthesia was applied (100 μl xylazine andketamine solution IM) and the hair overlaying the tumor was clipped andshaved. After the treatment, the rats were maintained in separate cagesand still fed with the same high fat diet. The rats were observed dailyand the morphological measurements of tumors—both laser treated anduntreated controls—were made twice a week.

3. Sensitizer Administration

The sensitizer indocyanine green (ICG) (Sigma Chemical Co., St. Louis)was used in two forms: ICG in water and ICG in glycated chitosan, theconcentration being 0.5% and 1% (g/100 ml) ICG to water or chitosan. Inthe case of chitosan solution, an ICG powder was mixed with glycatedchitosan made as hereinabove described, after the gel was brought to 37°C. from frozen state (−4° C.), in a glass grinder to obtain a uniformsolution. The sensitizer solution then was injected into the center ofthe targeted tumor, either 24 hours prior to laser treatment or just 10minutes before the procedure. The dosage varied between 70 μl to 400 μlto one tumor.

For the rats with transplanted primary tumors in both inguinal areas,only one tumor received the ICG-Chitosan injection. However both tumorswere treated using the same laser parameters.

4. Laser Treatment

The laser energy was delivered to the treatment sites through fibershaving diameters of 600 or 1200 μm. The tip of the fiber was maintained4 mm from the skin. The fiber tip was moved evenly and slowly throughall sides of the tumor to ensure a uniform energy distribution. A thinwater film (20° C.) was constantly applied on the surface of treatmentsites to prevent unnecessary damage of skin due to the surface heatbuild up.

For the rats with two simultaneous primary tumors, one tumor was lasedwith the aid of ICG-Chitosan and the other was treated by the laseronly. Fifty-six rats were treated using various laser parameters inconjunction with ICG-Chitosan solutions. Sixteen rats were injected with100-200 μl of a 1% ICG-H₂O solution and lased with various irradiancesfrom 3-5 minutes and from 3-10 watts.

Results:

1. The Survival Rates

In total, one hundred five (105) rats were used in the study up to Apr.1, 1995. All the rats were injected with the metastatic transplantablerat mammary tumor cells, either in one inguinal region or both. The ratswere divided into three groups: (1) control, (2) laser treated withICG-H₂O solution, and (3) laser treated with ICG-Chitosan solution. Thesurvival rates of the various grouped subjects are summarized on TableI.

TABLE I The Survival Rate of Rats with Tumor Transplant Group SurvivalDays Number of Rats Control 32.4 ± 3.6 13 Laser Treated 29.0 ± 3.5  7with ICG only Laser Treated  42.7 ± 28.9 55 with ICG-ChitosanICG-Chitosan Treated 32.9 ± 5.7 49 (w/o long survival rats) ICG-ChitosanTreated 114.3 ± 39.8  6 (long survival rats only)* ICG-Chitosan Treated33.6 ± 6.0 36 5w @ 3 to 6 mins (w/o long survival rats)** ICG-ChitosanTreated  45.4 ± 32.4 42 5w @ 3 to 6 mins (with long survival rats) *Outof the six long term survival rats, three are still alive. The datacollected in this table were up to January 16, 1995. **The six longsurvival rats were treated using this laser power and the duration range(see Table II for more detail).

The average survival time for the 33 control rats was 31.5 days afterthe tumor transplant; the average was 29.0 days for laser treated ratsinjected with aqueous ICG only; the overall average survival time was45.6 days for the ICG-Chitosan injected laser treated rats. Among the 56rats in the last group, six rats achieved long term survival (at leasttwice that of the control rats), without which the group survival ratewould be 32.8 days. On the other hand, the six rats gave rise to anaverage 152.0 days of survival. It is worthwhile to note that threerats, Srat3, Srat4 and Srat6, were still alive when the report waswritten after they have survived 220, 192 and 147 days, respectively, upto Apr. 1, 1995.

2. The Tumor Responses to Laser Treatment

Table I shows that tumor rats in both group 2 and group 3 respondedpositively to laser energy. Almost all the tumors had temperatureelevation immediately after the laser treatment. The ICG-H₂O orICG-Chitosan solution injected tumors usually raised temperature by 40°F., while the ICG free tumor still raised temperature but at a lowerlevel, usually 20° to 30° F. above the body temperature depending on thelaser power and duration.

Internal explosions often occurred during the procedure due to thesudden temperature increase. It was more evident under high laser power,above 10 watts for example. High power produced skin damage in mostcases, even with constant application of water droplets on the treatmentsurface.

The tumor cell destruction under high laser power (10-15W) was rathersuperficial and deeper tumor cells often survived the laser assault.Better results were obtained when lower laser powers were applied. Allthe six long term survival rats resulted from the 5 watt treatment (4rats with 3 minute exposure times and 2 rats with 5 minutes). See TableII.

TABLE II Parameters for Laser-ICG-Chitosan Treated Long Term SurvivalRats Survival ICG-Chitosan Administration Laser Treatment* Post TumorAnimal Tumor Transplant Time Prior to Power Duration Transplant RatNumber Date Locations Treatment Dosage Location Date (Watts) (minutes)(Days) Srat 1 3-18-94 Both Legs 24 hours 70 μl @ 1% Left Leg 3-29-945.00 3 156 Rat #5/3-29-94 Srat 2 5-23-94 Both Legs 24 hours 70 μl @ 1%Left Leg 6-3-94 5.00 3 125 Rat #1/6-3-94 Srat 3 8-19-94 Left Leg 24hours 150 μl @ Left Leg 9-1-94 5.06 5 150** Rat #5/9-1-94 0.5% as of1/16/94 Srat 4 9-16-94 Both Legs 24 hours 100 μl @ 1% Right Leg 9-27-945.20 5 120** Rat #2/9-27-94 as of 1/16/94 Srat 5 10-31-94 Left Leg 10minutes 150 μl @ Left Leg 11-8-94 4.95 3  65 Rat #3/11-8-94 0.5% Srat 610-31-94 Left Leg 10 minutes 100 μl @ Left Leg 11-11-94 5.12 3  80** Rat#4/11-11-94 0.5% as of 1/16/94 *Totally 42 rats were treated usingparameters in this range, resulting in a 14% survival rate. **Rat isstill alive.

In total, 43 rats were treated with laser power of 5 watts and anirradiation duration of 3 to 6 minutes; this gives rise to a 14% longterm survival rate in this group. Under these parameters, without thesix long survival rats, the average survival reached 32.8±6.3 days.

Immediately after laser treatment, all the tumors showed a slower growthwithin the first few days, then returned to a normal growth rate. Oftenthe tumors would be partially bitten or chewed, but that would not stopor slow the tumor growth. Most rats died around 30 to 35 days except forthe rats listed in Table II. About half the treated rats later developedsecondary tumors, most as “hand bags” (metastatic to lymph nodes in theaxillary region); the local expansions around the primary tumors werealso in lymph nodes. In either case, the secondary tumors continued togrow until death occurred, except for the rats in Table II.

3. Tumor Development of the Long Term Survival Rats

All six rats in Table II were treated using 5 watts power with eitherthree minutes duration (four rats) or five minutes (two rats). Moreimportantly, all of them were injected with ICG-Chitosan gel solution.For rats with two transplanted primary tumors, only one of the twotumors received the ICG-Chitosan solution and the other was lasedwithout any ICG sensitizer. For rats with only one primary tumor, theICG-Chitosan was used. The ICG-Chitosan solution was 0.5% to 1% and thedosage varied from 70 μl to 150 μl per tumor. The ICG-Chitosan wasusually injected directly into the tumor either 24 hours or 10 minutesbefore the laser treatment.

Like all the other rats, the tumors continued to grow after treatment,and most of the tumors metastasized along the milk line to the axillarynodes and the opposite inguinal nodes. However, the development oftumors took a dramatic turn at a later stage. Shown in FIGS. 1 and 2 arethe growth charts of the primary tumors of Srat1 and Srat2, treated bylaser; the left inguinal tumor was injected with ICG-Chitosan and theright inguinal tumors were not. The growth reached a peak around 50 daysafter the tumor transplant, and then started to regress. The tumorscontinued to shrink and reached their minimum size around 90 days.Afterwards the tumors started growing again, with Srat1 being moreaggressive than Srat2. Note the bigger burden on the right inguinal inboth cases (no ICG-Chitosan injection).

The secondary tumors of Srat1 and Srat2 appeared around day 20 aftertumor transplant, and went through the same pattern as theprimaries—growth/shrinkage/ growth, as shown in FIGS. 3 and 4. In thecase of Srat2, the recurrence of secondary tumors was almost negligible.

The tumor growth of three other rats is shown in FIG. 5. Srat3 and Srat6started with only one primary tumor and Srat4 with two. FIG. 5 shows amuch earlier response: tumor reduction started around 20 to 25 days.Furthermore, there are so far no secondary tumors, and the primarytumors have become only a small hard core of fibrous tissue; just aremnant of the tumor. Further development will be observed since allthree remain alive at this time (Apr. 1, 1995).

4. Improvement of the Experiment

Early experiments had been mainly focused on establishing workableconditions, including laser parameters, as well as the concentration anddosage of ICG-Chitosan. No long term survival was observed in the firstfew groups. Suggested by in vitro and in vivo results, work had beenproceeding in the laser power range of 3 to 5 watts. ICG-ChitosanChitosan solution between 0.5% to 1% seem to be effective. A steady longterm survival rate of 10% has been achieved, and even a 20% rate in oneof the recent rat groups.

Discussion

Clearly, the photothermal effect of the 808 nm diode laser on organizedtissue can be greatly enhanced when the chromophore ICG with anabsorption peak around 800 nm is used. The 808 nm laser energy canpenetrate readily through the normal tissue leaving the cellularstructure largely intact within regulated power ranges.

It is the ICG molecule, when injected to the target tissue, that absorbsstrongly the 808 nm radiation and reaches an excited state. When themolecule returns to ground state, the stored energy is released in theform of heat which can be absorbed by surrounding tissue to elevatetemperature. (The excited ICG molecule may also cause other biochemicalreactions which may be the key in our induced immunological responses.)When a sufficient amount of ICG molecules are excited within a certaintime (normally shorter than the tissue thermal relaxation time), thereleased heat can be absorbed by tissue cells faster than it can bedispersed. If the exposure to laser is long enough, the accumulated heatenergy can raise tissue temperature to a level at which photothermaldestruction of organized tissue can occur. This destruction can beachieved with certain selected laser powers and irradiation durations.It appears that a laser power around 3 to 5 watts is sufficient to causefatal injury to tumor cells.

Higher powers, in conjunction with ICG, can cause quick and more drasticthermal injury to the tissue, but some undesirable results may arise.The high power irradiation gives rise to a much faster temperaturebuild-up, often leading to internal explosions and quick tissuecarbonization, particularly on the treatment surface when oxygen isabundant in the air. A surface cooling procedure, either by water filmor by helium gas may not be able to slow down the carbonization, whichchanges the surface tissue properties from almost transparent to highlyabsorbent to the 808 nm wavelength. The 808 nm radiation would be inturn absorbed further by the carbonized tissue. This would impede thepenetration of the laser energy, resulting in a superficial and limitedspatial thermal destruction of intended malignant tissue.

The thermal impact alone may slow down the short term tumor growth, butmay not alter the predestined fate for hosts who have acquired tumors.As shown in Table I, the second group, treated by laser with the aid ofICG-H₂O solution, did not show any improvement on the survival rate. Ofcourse, the destruction of tumor cells due to photothermal interactionwas a predominant effect. However, due to the aggressive nature of thetumors, total eradication was rarely achieved by the thermal destructionalone; just as in the cases of surgical removal and radiation therapy.

Conclusion

It is thus apparent that other mechanisms must be utilized in order todeal with the root cause of the malignant cell multiplication. The idealmechanism, of course, is the self-immunological defense system, whichcan prevent growth of abnormal cells. It is when the natural immunefunctions are debilitated, or not adequate in response to foreignelement invasion, that cancers occur. If natural immune defense failedto stop the uncontrolled growth of cells, endogenous or exogenous,stimulated immune responses are needed. As previously stated, it haslong been known that certain chemicals may enhance the natural defensemechanism, but often the enhancement is one or several steps behind thetumor growth. The model described herein was directed to a combined PDTand immunotherapeutic treatment.

Other modalities, such as chemotherapy and radiation therapy, often killcells indiscriminately so that the collateral damage can be just asfatal. And though PDT relies on heat and/or toxic singlet oxygengenerated by treatment of laser and certain photosensitizers to killtumor cells, it still is not a means to affect the host'sself-immunological defense system, other than just incidentally. Byusing a combination of laser, chromophore, and immunomodulator, a novelcancer treatment is provided.

The effect of this invention in stimulating cell-mediated and humoralimmune responses in the host is shown FIGS. 1-5. The growth charts(FIGS. 1 and 2) show the change of the tumor burden. Fifty days aftertumor transplantation (40 days after laser-ICG-Chitosan treatment),primary tumors reached their maximum sizes. The reduction of tumorgrowth and size afterwards can only be explained by the generatedimmunological response, since only the self-defense mechanism, whenfully developed, would slow down and stop the tumor growth, and thedisfunctioning tumor cells would be engulfed by macrophages. Withoutthis exotic feature, none of the rats could have survived over 35 or 40days, as demonstrated by the first two groups of experimental rats inTable I.

The ICG-Chitosan injected tumor (left inguinal in most cases of the sixrats in Table II) had a slower growth rate, a sign of a more directimpact of the immunoadjuvant, even though both tumors were treated withsame laser power and duration. The ICG-Chitosan injected tumor ingeneral responded more to the laser treatment. The thermal interactionalone, in conjunction with ICG, had effectively reduced the tumor burdenon a large scale. The chitosan apparently added the immunologicalstimulation. The combination of the thermal and immunological effectsappear to be the explanation as to why the left inguinal tumors had lessgrowth than those of the right as shown in FIGS. 1 and 2.

The generation and acceleration of the immunological defense systemresponse is further supported by the evolution of the secondary tumorsof the long surviving rats. The metastasis usually occurred to half therats around 15 to 20 days after the transplantation of primary tumors.The secondary tumors appeared in most cases along the milk lines andcontinued to grow until death. However, the metastatic tumors of Srat1and Srat2 in FIGS. 3 and 4 showed exactly the same trend as in theprimaries (FIGS. 1 and 2), with neither ICG-Chitosan injection nor lasertreatment. FIG. 5 depicts the growth of primary tumors of three rats(Srat3, Srat4 and Srat6), all of them following the samedevelopment—growth/treatment/growth reduction. Furthermore, these threerats developed their full responses earlier; the tumor growth wasstopped around 20 to 25 days after tumor transplantation and secondarytumors never appeared. This early establishment of the inducedimmunological defense mechanism may explain why these rats are stillalive and have no signs of tumor recurrence.

In conclusion, long term survival with total cancer eradication can beachieved by laser-chromophore-adjuvant induced immunological responses.It is a combined result of reduced tumor burden due to photothermalinteractions and an enhanced immune system response due to the additionof chitosan or other immunomodulators. The experimental results havebeen improving constantly. The first few groups yielded no long termsurvivors whereas a steady 10% long term survival rate is now achieved.Within the current adapted laser power (5 watts) and the irradiationduration (3 to 6 minutes), the long term survival rate reaches up to14%.

B. Alternative Physical Therapies

Nonthermal cytotoxic phototherapy, as described above, usually involvesthe systemic administration of tumor localizing photosensitizingcompounds and their subsequent activation by laser. Upon absorbing lightof the appropriate wavelength the sensitizer is converted from a stableatomic structure to an excited state. Cytotoxicity and eventual tumordestruction are mediated by the interaction between the sensitizer andmolecular oxygen within the treated tissue to generate cytotoxic singletoxygen.

Neoplastic cellular destruction, however, can also be achieved bysubjecting the neoplasm to heat energy. Possible mechanisms for thermalcell death include direct damage to DNA, cell membrane damage, the heatstimulated production of special proteins, and disruption of themicrovasculature of the tumor. See S. G. Brown, MD, Phototherapy ofTumors, World Journal of Surgery, Vol. 7, pp. 700-709 (1983). Thethermal effects of photocoagulation and cellular vaporization producedby hyperthermia are believed to be sufficient to induce neoplasticcellular destruction and to generate fragmented neoplastic tissue andcellular molecules such that, when in the presence of an immunoadjuvant,would form an in situ vaccine as contemplated by the present invention.Means of inducing cellular hyperthermia include the use of lasers,ultrasound, microwaves, radio frequency induction or electric currents.

One laser treatment modality is that of stereotaxic interstitial lasertherapy (ILT). Interstitial laser photocoagulation or vaporizationdepends on the bare end of a laser fiber being inserted into a tumormass. The laser light is then absorbed as heat with the production of aregion of necrosis around the fiber tip. This is, thus, an invasivetechnique. The use of ILT in the treatment of breast cancer wasdescribed by Dowlatshahi, et al. in Stereotaxic Interstitial LaserTherapy of Early Stage Breast Cancer, The Breast Journal, Vol. 2, No. 5,pp. 305-311 (1996) and by Harries, et al. in InterstitialPhotocoagulation as a Treatment for Breast Cancer, British Journal ofSurgery, Vol. 81, pp. 1617-1619 (1994), both publications beingincorporated herein by reference.

Typically, ILT implores the use of a laser, such as an 805 nm diodelaser and a light transmissive fiber, such as a 400 μm quartz fiber.Under general or local anesthesia a needle is inserted into the tumorand the tip of the needle is positioned within the center of the tumorusing ultrasonography to monitor the needle position. The laser fiber isadvanced through the canula of the needle and the needle is thenwithdrawn slightly so that the tip of the fiber lays within the tumor.The tumor is then treated with laser light at a power and for anexposure time sufficient to cause neoplastic cellular destruction.Thermocouples may be used to display the intratumor temperature in realtime. Apparatae and methods for conducting ILT are described in U.S.Pat. Nos. 5,169,396; 5,222,953 and 5,569,240, which patents areincorporated herein by reference.

Tumor hyperthermia can also be achieved by treating a tumor with highintensity ultrasound. Ultrasound is a penetrating, directional and evenfocusable radiation. High energetic focused ultrasound for therapeuticuse involves using flexible ultrasound equipment for tissue destructionby generating small hot spots with one or more focused transducers. Thiscould be accomplished, depending upon tumor location, as an invasive ornon-invasive technique. The biological effect of ultrasound involvesboth heat and cavitation. A coupling medium may be used to transmit theultrasonic beam(s) to tissue.

Both invasive and non-invasive techniques of applying heat energy havebeen developed for microwave and radio frequency heating. With radiofrequencies the non-invasive invasive applicators are either capacitiveplates or inductive coils. With microwaves radiative apertures are used.Invasive applicators include the use of a radiating monopoles formicrowaves and RF needle electrodes or implanted ferromagnetic seeds(selectively heated by external inductive coils) for radio frequencies.The penetration and severity of damage can be controlled by thefrequency and total energy of the applied radiation. The volume of tumorthat can be heated is comparable to that heated using a Nd:YAG laserwith the transmission fiber inserted into the tumor (radius of 1-2 cmfrom the treatment point). The radiating monopole is a miniature coaxialtransmission line which can be made flexible and implanted surgically orinserted via body orifices. See S. G. Brown, Phototherapy of Tumors,supra.

One other potential way of causing neoplastic cellular destruction byhyperthermia would be to circulate heated water about the tumor in orderto raise the temperature of the tumor. This could be done on peripheraltumors that are approachable with a device from the outside, but thisapproach might not be too effective at high temperatures.

Cryotherapy may also be used to achieve neoplastic cellular destruction.Cryotherapy is the use of extreme cold to destroy cancer cells.Cryotherapy was first used in treating external tumors, such as those onthe skin, by contacting the skin with liquid nitrogen. For internaltumors a cryoprobe can be used to circulate liquid nitrogen into contactwith the tumor. Ultrasonography may be used to guide the cryoprobe andmonitor the freezing of the tumor mass. Cryotherapy has heretofore beenused in the treatment of, among other things, prostate cancer and livermetastases. It is sometimes used in combination with other cancertreatments such as radiation, surgery, and hormone therapy. As withother mechanisms of tumor destruction, the focus is on destroying thetumor without damaging nearby healthy tissue.

It is thought that several processes contribute together to achievecryosurgical cellular destruction. The creation of intracellular ice byrapid temperature loss is fatal to the cells. Moreover, as ice formsaround a cell the free water inside the cell is drawn off shrinking thecell and collapsing the walls or membranes inside the cell, releasingproteins or chemicals which can be toxic. In addition, as ice whichsurrounds shrunken cells begins to thaw, large amounts of free waterproduced by the thawing ice will rush back inside the cells, causingthem to burst. Thus, it is believed by some that the physical featuresof cryosurgery which are most important in producing extensive celldestruction include rapid freezing to very low (−195° C.) temperatures,and a slow thawing.

Cryotherapy has heretofore been used in treating prostate cancer, skindisorders and retinopathy in premature infants. As with hyperthermia,thermocouples can be used to monitor temperature to confirm that allareas of the tumor are properly frozen.

C. Special Products Derived from Tandem Therapies

Certain special products produced by the interaction of tumor antigensand the adjuvant with the host may be derived from the describedtherapies.

For example, one of these products would be specific antibodies directedagainst certain antigenic domains found on the cell surface. When theantibodies and antigens meet in the circulation a complex is formed thatcan be separated and identified. The separated products then can be usedas probes to determine if the antigens are common to all human tumors ofthe same type.

Likewise, free antibodies can be used to collect circulating freeantigens for identification and possible synthesis to form the basis ofa screening test for very early breast cancer.

It is also possible for these antibodies to be used to locate and treatrecurrent or metastatic tumors in areas not approachable by the originaltechnique. This is accomplished by tagging the antibody with chemicalsthat aid in localization or drugs that are toxic to cells.

Thus, a large variety of products could be generated from the reactionof the host to the products of the described treatment. Likewise theseproducts may be keys to other methods to attack tumor cells, bothdirectly and indirectly.

While the invention has been described with a certain degree ofparticularity, it is manifest that many changes may be made in themethod hereinabove described without departing from the spirit and scopeof this disclosure. It is understood that the invention is not limitedto the embodiments set forth herein for purposes of exemplification, butis to be limited only by the scope of the attached claim or claims,including the full range of equivalency to which each element thereof isentitled.

What is claimed is:
 1. A method for generating an in situ autologousvaccine in a host possessing a neoplasm, comprising the steps of: (a)localizing an immunoadjuvant in said neoplasm to obtain a conditionedneoplasm; and (b) subjecting said conditioned neoplasm to directphysical stress sufficient to induce neoplastic destruction in saidconditioned neoplasm so as to generate fragmented neoplastic tissue andcellular molecules; said in situ vaccine comprising an amalgam of saidfragmented tissue and cellular molecules physically associated with saidimmunoadjuvant.
 2. The method according to claim 1, wherein step (b)comprises inducing neoplastic destruction in said conditioned neoplasmby hyperthermia.
 3. The method according to claim 2, wherein step (b)comprises inducing photothermal neoplastic destruction in saidconditioned neoplasm.
 4. The method according to claim 3, wherein step(b) comprises irradiating said conditioned neoplasm.
 5. The methodaccording to claim 4, wherein said conditioned neoplasm is irradiatedwith a laser.
 6. The method according to claim 5, wherein step (b)comprises non-invasively irradiating said conditioned neoplasm.
 7. Themethod according to claim 1, wherein step (b) comprises inducingneoplastic destruction in said conditioned neoplasm by cryotherapy.